As human retinas are rich in auto fluorescent compounds, such as lipofuscin, it is not practical to use immunofluorescence methods to localize molecules in tissue sections during basic eye research or in clinical evaluations of eye pathology;this is especially true for the retinal pigment epithelium. Immunolocalization within human retinas is limited to old-fashioned histochemical protocols, which are characterized by low resolution, low sensitivity, and poor contrast. We hypothesize that endogenous auto fluorescence in human retinal sections can be greatly reduced or eliminated by taking advantage of certain physical properties of inorganic nanoparticles containing rare earth lanthanides. Our objective in testing this hypothesis is to establish novel techniques to study the human retina based upon the use of lanthanide nanoparticles. In the first aim, human retinal tissue sections will be labeled with first step antibodies directed against retinal antigens, followed by labeling with a second-step reagent covalently linked with nanoparticles containing lanthanides (Eu3+ or Tb3+), which exhibit a very large Stokes shift (~300 nm) and fluorescent lifetimes of 600 microseconds. Using a flash-lamp and a time-gated trigger in conjunction with a temporally modulated camera, we will collect only the longer-lived lanthanide emission. To test this method's utility, a panel of antibodies reactive with different portions of the retina will be examined in pathology samples from control and diabetic retinopathy patients. In the second aim, we will utilize samples labeled with first step antibodies followed by antibodies tagged with nanoparticles containing up-converting phosphors (UCPs). Anti-Stokes microscopy will be used to image these nanoparticles on retina without confounding auto fluorescence. Excitation will be provided at 980 nm to promote anti-Stokes or "up-converted" green fluorescence emission of Er3+-containing UCPs. By using infrared excitation, no excitation of endogenous auto fluorescent species in the retina will take place because: the retina does not absorb this infrared light and the retina does not display the unique physical properties of Er3+ (long fluorescence lifetime and anti-Stokes emission). We anticipate that these innovative immunofluorescence microscopy methods will be broadly used in ophthalmology and in the clinical practice of ocular pathology. Moreover, these studies will lay the groundwork for a new generation of human retinal biology studies that were previously obscured by auto fluorescence, such as receptor and channel localization, enzyme translocation, oxidant damage, and protein-protein interactions, among others. PUBLIC HEALTH RELEVANCE: This research program will lead to the development of new methods to conduct eye research and ocular pathology. Consequently, it will likely contribute to new discoveries in eye research as well as improved patient care.